Biotinylation liposomes

Biotinylated liposomes usually are created by modification of PE components with an amine-reactive biotin derivative, for example NHS-LC-Biotin (Chapter 11, Section 1). The NHS ester reacts with the primary amine of PE residues, forming an amide bond linkage (Figure 22.19). A better choice of biotinylation agent may be to use the NHS-PEG -biotin compounds (Chapter 18), because the hydrophilic PEG spacer provides better accessibility in the aqueous environment than a hydrophobic biotin spacer. Since the modification occurs at the hydrophilic end of the phospholipid molecule, after vesicle formation the biotin component protrudes out from the liposomal surface. In this configuration, the surface-immobilized biotins are able to bind (strept)avidin molecules present in the outer aqueous medium. 

Figure 22.18 Biotinylated liposomes may be used in immunoassay systems to enhance the signal for detection or measurement of specific analytes. The liposome components may be constructed to include fluorescent molecules to facilitate detection of antigens within tissue sections.

The following method for the formation of a biotinylated liposome is adapted from Plant et al., 1989). It assumes prior production of B-PE. 

Prepare a biotinylated liposome construct by first dissolving in chloroform, the lipids DMPC cholesterol dicetylphosphate (Sigma) at mole ratios of 5 4 1, and adding to this solution 0.1 mol percent B-PE. Larger mole ratios of B-PE to total lipid may result in nonspecific aggregation of liposomes in the presence of avidin. Maintain all lipids under an inert atmosphere to prevent oxidation. 

Figure 22.19 Biotinylated liposomes may be formed using biotinylated PE. Reaction of NHS-LC-biotin with PE results in amide bond linkages and a long spacer arm terminating in a biotin group.

The biotinylated liposomes prepared by this procedure may be stored under an inert-gas atmosphere at 4°C for long periods without degradation. 

Figure 22.21 Antibodies may be conjugated to liposomes using an indirect approach incorporating a (strept)avidin-biotin system. Biotinylated liposomes may be complexed with biotinylated antibodies using (strept)avidin as a bridging molecule or may be complexed with an antibody-(strept)avidin conjugate.

Hashimoto, K., Loader, J.E., and Kinsky, S.C. (1986) Iodoacetylated and biotinylated liposomes Effect of spacer length on sulfbydryl ligand binding and avidin precipitability. Biochim. Biophys. Acta 856, 556-565. 

Fig. 2. A Interaction of biotin with the tetrameric protein streptavidin. B Schematic representation of the aggregation of biotinylated liposomes induced by the tetrameric streptavidin protein (taken from [16])...

An avidin-biotin system has been used to attach antibodies in the bilayer of DDSs. Xiao et al. developed a three-step strategy to improve the tumor-to-tissue ratio of anticancer agents [184], Two antibodies specific for the CA-125 antigen that is highly expressed on NIH OVCAR-3 cells were used. These cells were prelabeled with biotinylated anti-CA-125 antibody and fluoroscein isothiocyanate (FITC)-labeled streptavidin (SAv) prior to administration of biotinylated liposomes. Both antibodies were specifically bound to the cell surface of OVCAR-3 cells but not to SK-OV-3 cells, which do not express the specific antibody. Antibody biotinylation did not affect its immunoreactivity. 

Soininen SK, Lehtolainen-DaUdlic P, Karppinen T, Puustinen T, Dragneva G, Kaikkonen MU, et al. Targeted delivery via avidin fusion protein intracellular fate of biotinylated doxorubicin derivative and cellular uptake kinetics and biodistribution of biotinylated liposomes. EurJ Pharm Sci 2012 47 848-56. 

Figure 15 (A) Faradaic impedance spectra of (a) The (17)-functionalized An electrode, (b) After interaction of the sensing electrode with (18) (5 X 10" M), which was pretreated with (20) (1 X 10 M, 30 min, 25°C). (c) After treatment of the resulting electrode with avidin (2.5 pg-mL" ). (d) After interaction with the biotinylated liposomes, (21). (e) After treatment of the interface for a second time with avidin (2.5 0,g mL" ), (f) After interaction of the interface for a second time with the biotinylated liposomes, (21). Data were recorded in 0.1 M phosphate buffer, pH = 7.2, in the presence of [Fe(CN)g] " , (1 1), as redox-probe. (B) Calibration curve corresponding to the changes in the electron transfer resistances of the sensing electrode upon interaction with the analyte DNA, (18), at different concentrations and enhancement of the sensing process by a double-step avidin/ biotinylated liposome amplification path. corresponds to the difference in the electron transfer resistance after a double-step avidin/biotinylated liposome amplification and the electron resistance of the (17)-functionalized electrode.

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